Abstract. Brainswellingisaseriouscomplicationofmultiplediseaseconditionsincludingliverfailure,metastatic tumors,traumaticbraininjuryandischemicstroke.TBIandstrokeafflict1.4Mand700Kpersonsperyearin theUSalone,andisacostlyhealthcareburdenandadevastatingsocialburden.Currenttreatmentsfor brainswellingarelimitedandgenerallyineffective,highlightingthedramaticunmetneedforbetter therapeutics.Abetterunderstandingofthemolecularpathwaysandcellularmechanismsissorelyneeded toidentifynewdrugtargetsaswellasmorepredictivebiomarkersthatcanstratifypatientsforclinical treatmentdecisions.Thegoalofthisprojectistoidentifynewdrugtargetsandbiomarkersofresponsefor cytotoxicedemaofastrocytes.Weproposetouseanovelandinnovativetechnologythatwehave developedthatwilltakeanunbiasedapproachtofunctionallyidentifyingthecausalmediatorsofastrocyte swelling.Ourapproachusesalargepanelofgeneticallydiverseastrocytelinestoidentifythegenesand pathwaysthatmechanisticallyunderliecytotoxicedema.InPhaseI,wewilldeveloptwohighthroughput kineticassaysforastrocyteswellingthatarerobust,scalableandautomatableforscreeningcompounds thatinduceorblockswelling.Animpedancebasedmorphologicalassaywillmeasureswellingand recovery,andarapidcalciumfluxassaywillmeasurecationicinflux.InPhaseIIwewillusetheseassays toscreen~300geneticallydiverseastrocytelines,andthenmapandidentifythegenesthatmediate responsetoinducersandblockersofcellswelling.Validationofcandidatetargetgeneswillbeconductedin bothhumanandmouseastrocytes.Humangenesthatmodifyhumanastrocyteresponsetocompoundare theultimateaimsandend-productsofthisproject.Thosegenesandsequencevariantsinthehuman populationwillalsobeevaluatedaspotentialprognosticbiomarkersusingexistingclinicaldatain retrospectiveanalyses.